Plant cell walls consist mainly of the large biopolymers cellulose, hemicellulose, lignin, and pectin. Cellulose and hemicellulose constitute an important renewable and inexpensive carbon source for the production of fermentable sugars. Cellulose consists of D-glucose units linked together in linear chains via beta-1,4 glycosidic bonds. Hemicellulose consists primarily of a linear xylan backbone comprising D-xylose units linked together via beta-1,4 glycosidic bonds and numerous side chains linked to the xylose units via glycosidic or ester bonds (e.g., L-arabinose, acetic acid, ferulic acid, etc.).
The term lignocellulose is commonly used to describe plant-derived biomass comprising cellulose, hemicellulose and lignin. Much attention and effort has been applied in recent years to the production of fuels and chemicals, primarily ethanol, from lignocellulose-containing feedstocks, such as agricultural wastes and forestry wastes, due to their low cost and wide availability. These agricultural and forestry wastes are typically burned or landfilled; thus, using these lignocellulosic feedstocks for ethanol production offers an attractive alternative to disposal. Yet another advantage of these feedstocks is that the lignin byproduct, which remains after the cellulose conversion process, can be used as a fuel to power the process instead of fossil fuels. Several studies have concluded that, when the entire production and consumption cycle is taken into account, the use of ethanol produced from cellulose generates close to zero greenhouse gases.
In comparison, fuel ethanol from feedstocks such as cornstarch, sugar cane, and sugar beets suffers from the limitation that these feedstocks are already in use as a food source for humans and animals. A further disadvantage of the use of these feedstocks is that fossil fuels are used in the conversion processes. Thus, these processes have only a limited impact on reducing greenhouse gases.
Lignocellulosic biomass has also been considered for producing other fermentation products besides ethanol. Examples of such products include lactic acid, sorbitol, acetic acid, citric acid, ascorbic acid, propanediol, butanediol, xylitol, acetone, and butanol.
The first chemical processing step for converting lignocellulosic feedstock to ethanol or other fermentation products involves hydrolysis of the cellulose and hemicellulose polymers to sugar monomers, such as glucose and xylose, which can be converted to ethanol or other fermentation products in a subsequent fermentation step. Hydrolysis of the cellulose and hemicellulose can be achieved with a single-step chemical treatment or with a two-step process with milder chemical pretreatment followed by enzymatic hydrolysis of the pretreated lignocellulose with cellulase enzymes.
In a single-step chemical treatment, the lignocellulosic feedstock is contacted with a strong acid or alkali under conditions sufficient to hydrolyze both the cellulose and hemicellulose components of the feedstock to sugar monomers.
In a two-step chemi-enzymatic hydrolysis process, the lignocellulosic feedstock is first subjected to a pretreatment under conditions that are similar to, but milder than, those in the concentrated acid or alkali hydrolysis process. The purpose of the pretreatment is to increase the cellulose surface area and convert the fibrous feedstock to a muddy texture, with limited conversion of the cellulose to glucose. If the pretreatment is conducted with acid, the hemicellulose component of the feedstock is hydrolyzed to xylose, arabinose, galactose, and mannose. The resulting hydrolysate, which is enriched in pentose sugars derived from the hemicellulose, may be separated from the solids and used in a subsequent fermentation process to convert the pentose sugars to ethanol or other products. If the pretreatment is conducted with alkali, very little hydrolysis of the polysaccharides occurs; however, the alkali treatment opens up the surface of the lignocellulose by reacting with acidic groups present on the hemicellulose.
After the pretreatment step, the cellulose is subjected to enzymatic hydrolysis with one or more cellulase enzymes such as exo-cellobiohydrolases (CBH), endoglucanases (EG) and beta-glucosidases (beta-G). The CBH and EG enzymes catalyze the hydrolysis of the β-1,4-D-glucan linkages in the cellulose. The CBH enzymes, e.g., CBHI and CBHII, act on the ends of the glucose polymers in cellulose microfibrils and liberate cellobiose, while the EG enzymes act at random locations within the cellulose polymer. Together, the cellulase enzymes hydrolyze cellulose to cellobiose, which, in turn, is hydrolyzed to glucose by beta-G. In addition to the CBH, EG, and beta-G enzymes, other enzymes or proteins that enhance the enzymatic degradation of the pretreated lignocellulosic substrate may be present during the hydrolysis reaction, e.g., xylanases, beta-xylosidases, beta-mannanase, acetyl xylan esterases, ferulic acid esterases, swollenins, and expansins. The presence of xylanases may be advantageous, for example, in cases where significant amounts of xylan are present in the pretreated feedstock.
If the pentose sugars are separated from the solids between the pretreatment and enzymatic hydrolysis steps, glucose will be the main sugar monomer in the hydrolysate produced by the enzymatic treatment. If the pentose sugars released by the chemical pretreatment step are carried through to the enzymatic hydrolysis step, the hydrolysate will contain glucose and xylose in about a 2:1 weight ratio, with L-arabinose being about 3-5 wt % of the total sugar monomers. Conversion of the hexose and pentose sugars in the resulting lignocellulosic hydrolysate (sometimes referred to as a lignocellulose hydrolysate) to ethanol or another product(s) is carried out in a subsequent microbial fermentation.
If glucose is the predominant sugar present in the hydrolysate, the fermentation is typically carried out with a Saccharomyces spp. yeast, which converts this sugar and other hexose sugars present into ethanol. However, if the hydrolysate comprises a significant proportion of pentose sugars, such as xylose and arabinose derived from hemicellulose, the fermentation is carried out with a microbe that naturally possesses, or has been engineered to possess, the ability to ferment xylose and/or arabinose to ethanol or another product(s).
Examples of microbes that can naturally grow on and/or ferment pentose sugars, such as xylose or arabinose, to ethanol or sugar alcohols include, but are not limited to, certain species of yeasts from the genera Candida, Pichia, and Kluyveromyces. However, such yeasts typically ferment glucose at a much slower rate than Saccharomyces. This is a particularly significant limitation in a process for fermenting lignocellulosic hydrolysate containing large proportions of glucose and xylose.
Examples of microbes that have been genetically modified to utilize xylose for growth or fermentation include, but are not limited to, recombinant Saccharomyces strains into which has been inserted either (a) the xylose reductase (XR)-encoding and xylitol dehydrogenase (XDH)-encoding genes (XYL1 and XYL2, respectively) from Pichia stipitis (U.S. Pat. Nos. 5,789,210, 5,866,382, 6,582,944, and 7,527,927, and European Patent No. 450530) or (b) a fungal or bacterial xylose isomerase (XI) gene (U.S. Pat. Nos. 6,475,768 and 7,622,284). Such strains are able to utilize both xylose and glucose for growth and/or fermentation. Examples of yeasts that have been genetically modified to utilize L-arabinose for growth and/or fermentation include, but are not limited to, recombinant Saccharomyces strains into which genes from either fungal (U.S. Pat. No. 7,527,951) or bacterial (International Patent Pub. No. WO 2008/041840) arabinose metabolic pathways have been inserted. In some instances, recombinant Saccharomyces strains have been developed to utilize both xylose and arabinose for growth and/or fermentation (International Patent Pub. No. WO 2006/096130). Further genetic modifications to such strains have been made, by genetic engineering and/or adaptive evolution techniques, to enhance the xylose conversion rate or ethanol yield from xylose. These modifications include overexpression of sugar transporters (U.S. Patent Pub. No. 2007/0082386), deletion of endogenous nonspecific aldose reductase GRE3 (U.S. Pat. No. 6,410,302), and enhancement in the pentose phosphate pathway (International Patent Pub. No. WO 2005/108552; U.S. Patent Pub. Nos. 2006/0216804 and 2007/0082386). However, whereas the xylose conversion rates and/or yields of ethanol from xylose were increased with pure sugar fermentations, similar results were either not observed or not reported for fermentations of pentose sugars in lignocellulosic hydrolysates.
Lignocellulosic hydrolysates, regardless of whether produced by a single-step chemical treatment process or by a two-step chemical pretreatment and enzymatic hydrolysis process, typically comprise not only sugar monomers such as glucose, xylose and arabinose, but also lignin monomers, acetate (released from the hemicellulose side-chains), and chemical reaction products of the sugars, such as furfural (from xylose) and hydroxymethylfurfural (from glucose). Acetate, furfural, and hydroxymethylfurfural are well-known inhibitors of microbial growth and/or fermentation processes converting sugars to ethanol. The presence of acetic acid in lignocellulosic hydrolysates is especially problematic as it inhibits yeast cell growth and thus can significantly reduce the yield of fermentation products (Abbott et al. (2007) FEMS Yeast Res. 7:819-33). Other yeast inhibitors that arise when converting lignocellulosic feedstocks to fermentable sugars are furfural and 5-hydroxymethylfurfural (HMF). Furfural and HMF result from the loss of water molecules from xylose and glucose, respectively, by exposure to high temperatures and acid. The inhibitory effects of these compounds decrease the efficiency of the fermentation operations by lengthening the time required for carrying out the fermentation, increasing the amount of yeast required, decreasing the final yields, or a combination of these.
It is possible to remove these inhibitors from the lignocellulosic hydrolysate prior to their conversion to ethanol (or other chemicals) in a yeast fermentation by physical separation methods. However, these processes are often costly and are likely to result in an increase in overall costs for the production of the ethanol or other desired fermentation product(s) from the lignocellulosic biomass.
For example, one method that has been proposed to reduce the concentration of inhibitors arising from hydrolysis of lignocellulosic feedstocks is overliming, which involves the addition of Ca(OH)2 to precipitate inhibitors from lignocellulosic hydrolysates, thereby improving the subsequent fermentation using yeast. Such processes are disclosed by U.S. Pat. Nos. 2,203,360; 4,342,831; 6,737,258; 7,455,997; and Wooley et al. (In: Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzyme Hydrolysis Current and Future Scenarios (1999) Technical Report, National Renewable Energy Laboratory, pp. 16-17). However, any handling of the lime cake is difficult and costly. In addition, the introduction of calcium into the stream increases the likelihood that calcium scale will deposit on evaporators, distillation columns, and other process equipment. The clean-up and avoidance of scale increases the cost of sugar processing.
Another method that has been proposed to remove inhibitors of fermentation is ion exchange. For example, ion exchange has been investigated by Nilvebrant et al. ((2001) App. Biochem. Biotech. 91-93:35-49) in which a spruce hydrolysate was treated to remove fermentation inhibitors, such as phenolic compounds, furan aldehydes, and aliphatic acids. U.S. Pat. No. 7,455,997 and Wooley et al. (supra) report the use of ion exchange to remove acetic acid from an acid-hydrolyzed mixture obtained from wood chips, followed by lime treatment. Similarly, Watson et al. ((1984) Enzyme Microb. Technol. 6:451-56) disclose the use of ion exchange to remove inhibitors, such as acetic acid and 2-furaldehyde (furfural), from a sugar cane bagasse acid hydrolysate prior to fermentation. Furthermore, Tran and Chambers ((1986) Enzyme Microb. Technol. 8:439-44) disclose various treatments to remove inhibitors prior to fermentation from an acid prehydrolysate from red oak, including mixed bed ion resin treatment.
In practice, several factors limit the effectiveness of ion exchange treatment to remove inhibitors. First, the multi-component nature of the streams results in an inefficient removal of some species at any single set of conditions. Second, the high ionic load demands very frequent and expensive regeneration of the resin. Finally, not all of the inhibitors are ionic, and ion exchange is ineffective in removing nonionic compounds from sugar.
U.S. Patent Pub. No. 2008/0171370 reports that gallic acid can be used to detoxify hydrolysates resulting from pretreating a lignocellulosic material by binding acetic acid. As disclosed therein, the gallic acid is a natural polymer comonomer, i.e., the core of the gallotannin structure, and therefore is a natural means to polymerize phenols and acetic acid in a Fischer esterification with a sulfuric acid catalyst.
International Patent Pub. No. WO 2008/124162 discloses the selective removal of acetate from a sugar mixture containing xylose and glucose by an E. coli strain that is able to convert acetate to a biochemical such as ethanol, butanol, succinate, lactate, fumarate, pyruvate, butyric acid, and acetone. The E. coli has been deleted in four genes that would otherwise code for proteins involved in xylose and glucose utilization—thereby preventing the consumption of either xylose or glucose by the E. coli—but that have no known effect on acetate metabolism. After acetate conversion to a biochemical, xylose and glucose fermentation are conducted on the sugar mixture using separate microorganisms, one with the ability only to ferment xylose, and the other with the ability only to ferment glucose. However, the process is not directed to removing unwanted sugars from a sugar hydrolysate, but rather to maximizing the conversion of all sugars present in the mixture to ethanol or other biochemicals.
An alternative approach to detoxification of the lignocellulosic hydrolysate is to develop yeast strains that are tolerant of the inhibitory compounds present in such hydrolysates. For example, adaptation to lignocellulose hydrolysates has been reported for Pichia and Saccharomyces strains (Huang et al. (2009) Bioresource Technol. 100:3914-20; Martin et al. (2007) Bioresource Technol. 98:1767-73). Although this approach generated strains with some tolerance to the hydrolysate, the genotype(s) of the adapted strains was not characterized.
Other attempts to characterize and/or improve tolerance to lignocellulose hydrolysates have been directed towards tolerance to the three major inhibitors in lignocellulosic hydrolysates—acetic acid, furfural, and hydroxymethylfurfural (HMF). For example, strains of Pichia and Saccharomyces have been adapted to media containing furfural and/or hydroxymethylfurfural (Liu et al. (2004) J. Ind. Microbiol. Biotechnol. 31:345-52.; Liu et al. (2005) Appl. Biochem. Biotechnol. 121-124:451-60). Other studies have attempted to identify genes that contribute to inhibitor tolerance by screening collections of yeast single-deletion strains to identify those deletions that increase the sensitivity of the yeast to furfural (Gorsich et al. (2006) Appl. Microbiol. Biotechnol. 71:339-49), acetaldehyde (Matsufuhi et al. (2008) Yeast 25:825-22), and lactic and acetic acids (Kawahata et al. (2006) FEMS Yeast Research 6:924-36). Expression profiling of a limited set of genes was conducted to determine changes in gene expression in a yeast strain adapted to furfural and HMF vs. a parental strain cultured in the presence of HMF (Liu et al. (2009) Mol. Genet. Genomics 282:233-44). However, neither the sensitivity of the resulting strains to, nor the expression profile of the strains cultured in the presence of, lignocellulose hydrolysates was reported.